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TWEAK-independent Fn14 self-association and NF-κB activation is mediated by the C-terminal region of the Fn14 cytoplasmic domain.

Brown SA, Cheng E, Williams MS, Winkles JA - PLoS ONE (2013)

Bottom Line: Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions.Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122.These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

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Related in: MedlinePlus

Transient Fn14-FL or Fn14-ΔEC overexpression, but not Fn14-ΔCT overexpression, can activate the NF-κB signaling pathway.(A) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔCT protein. The Fn14-FL construct is the same as shown in Fig. 2A but here the size of the cytoplasmic tail (CT) is indicated. The Fn14-ΔCT construct contains an Ig-κ chain signal peptide instead of the Fn14 signal peptide and an extra stretch of 9-aa residues that was introduced during the cloning procedure (denoted here as L for linker). The positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and grown for 24 hr. Cells were harvested, lysed, and equal amounts of protein were used for Western blot analysis using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (C) HEK293/NF-κB-luc cells were transfected in 6 well dishes with the indicated plasmids and grown for 24 hr. The cells were harvested, lysed, and NF-κB-luc reporter expression was assayed using a luminometer. Luciferase activity was normalized to the vector-transfected cells. The values shown are mean +/− SEM of triplicate wells from one representative experiment of three independent experiments. *P<0.01 versus vector using Student’s t test.
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pone-0065248-g003: Transient Fn14-FL or Fn14-ΔEC overexpression, but not Fn14-ΔCT overexpression, can activate the NF-κB signaling pathway.(A) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔCT protein. The Fn14-FL construct is the same as shown in Fig. 2A but here the size of the cytoplasmic tail (CT) is indicated. The Fn14-ΔCT construct contains an Ig-κ chain signal peptide instead of the Fn14 signal peptide and an extra stretch of 9-aa residues that was introduced during the cloning procedure (denoted here as L for linker). The positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and grown for 24 hr. Cells were harvested, lysed, and equal amounts of protein were used for Western blot analysis using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (C) HEK293/NF-κB-luc cells were transfected in 6 well dishes with the indicated plasmids and grown for 24 hr. The cells were harvested, lysed, and NF-κB-luc reporter expression was assayed using a luminometer. Luciferase activity was normalized to the vector-transfected cells. The values shown are mean +/− SEM of triplicate wells from one representative experiment of three independent experiments. *P<0.01 versus vector using Student’s t test.

Mentions: There is substantial experimental evidence supporting the proposal that when cellular Fn14 levels are high, Fn14 can activate downstream signaling events without ligand engagement (a process referred to as TWEAK-independent Fn14 signaling (reviewed in [4]). However, the studies performed to date have not ruled out the possibility that the Fn14-triggered cellular effects observed, for example, NF-κB pathway activation [32], [44], [45], could be mediated by a low level of soluble TWEAK in the cell culture medium (either present in FBS [37], [38] or secreted by the cells under investigation). The cloning of Fn14-ΔEC afforded us the opportunity to directly test whether ectopic Fn14 overexpression promoted TWEAK-independent Fn14 signaling. As our readout for Fn14 signaling we selected NF-κB pathway activation, since TWEAK:Fn14 engagement in diverse cell types has been shown to stimulate this intracellular pathway (reviewed in [4]). HEK293/NF-κB-luc reporter cells were transiently transfected with vector or expression plasmids encoding either Fn14-FL (our positive control), Fn14-ΔEC, or Fn14-ΔCT, an Fn14 mutant that is missing the C-terminal 18-aa residues of the 28-aa Fn14 cytoplasmic tail (Fig. 3A) and therefore cannot bind TRAFs and activate signaling pathways (our negative control) [44]. All Fn14 proteins had an N-terminal Myc epitope tag. Cells were harvested 24 h later and processed for Western blot analysis and luciferase reporter assays. The three Fn14 proteins were expressed at similar levels (Fig. 3B). We noted previously that Fn14 does not migrate at its predicted molecular mass when analyzed by SDS-PAGE under reducing conditions [1] and this is again illustrated on this particular blot. For example, the predicted molecular mass (MM) of the mature, 112-aa myc-tagged Fn14-FL protein is 12.1-kDa, but its apparent MM on this gel is ∼23-kDa. Also, the 101-aa Fn14-ΔCT mutant (11.0-kDa predicted MM) is running at a lower apparent MM than the much smaller, 77-aa Fn14-ΔEC mutant (8.4-kDa predicted MM). The basis for these findings is not known but protein amino acid composition and isoelectric point are two interrelated factors that can influence the mobility of proteins on SDS polyacrylamide gels. For small proteins like Fn14, the effects of these factors on electrophoretic mobility are even more apparent. One possible explanation for the anomalous migration of the three Fn14 species relative to one another is that the Fn14-FL and Fn14-ΔEC proteins are acidic (predicted pIs of 5.8 and 5.3, respectively) while the Fn14-ΔCT mutant is basic (predicted pI of 8.8).


TWEAK-independent Fn14 self-association and NF-κB activation is mediated by the C-terminal region of the Fn14 cytoplasmic domain.

Brown SA, Cheng E, Williams MS, Winkles JA - PLoS ONE (2013)

Transient Fn14-FL or Fn14-ΔEC overexpression, but not Fn14-ΔCT overexpression, can activate the NF-κB signaling pathway.(A) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔCT protein. The Fn14-FL construct is the same as shown in Fig. 2A but here the size of the cytoplasmic tail (CT) is indicated. The Fn14-ΔCT construct contains an Ig-κ chain signal peptide instead of the Fn14 signal peptide and an extra stretch of 9-aa residues that was introduced during the cloning procedure (denoted here as L for linker). The positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and grown for 24 hr. Cells were harvested, lysed, and equal amounts of protein were used for Western blot analysis using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (C) HEK293/NF-κB-luc cells were transfected in 6 well dishes with the indicated plasmids and grown for 24 hr. The cells were harvested, lysed, and NF-κB-luc reporter expression was assayed using a luminometer. Luciferase activity was normalized to the vector-transfected cells. The values shown are mean +/− SEM of triplicate wells from one representative experiment of three independent experiments. *P<0.01 versus vector using Student’s t test.
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Related In: Results  -  Collection

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pone-0065248-g003: Transient Fn14-FL or Fn14-ΔEC overexpression, but not Fn14-ΔCT overexpression, can activate the NF-κB signaling pathway.(A) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔCT protein. The Fn14-FL construct is the same as shown in Fig. 2A but here the size of the cytoplasmic tail (CT) is indicated. The Fn14-ΔCT construct contains an Ig-κ chain signal peptide instead of the Fn14 signal peptide and an extra stretch of 9-aa residues that was introduced during the cloning procedure (denoted here as L for linker). The positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and grown for 24 hr. Cells were harvested, lysed, and equal amounts of protein were used for Western blot analysis using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (C) HEK293/NF-κB-luc cells were transfected in 6 well dishes with the indicated plasmids and grown for 24 hr. The cells were harvested, lysed, and NF-κB-luc reporter expression was assayed using a luminometer. Luciferase activity was normalized to the vector-transfected cells. The values shown are mean +/− SEM of triplicate wells from one representative experiment of three independent experiments. *P<0.01 versus vector using Student’s t test.
Mentions: There is substantial experimental evidence supporting the proposal that when cellular Fn14 levels are high, Fn14 can activate downstream signaling events without ligand engagement (a process referred to as TWEAK-independent Fn14 signaling (reviewed in [4]). However, the studies performed to date have not ruled out the possibility that the Fn14-triggered cellular effects observed, for example, NF-κB pathway activation [32], [44], [45], could be mediated by a low level of soluble TWEAK in the cell culture medium (either present in FBS [37], [38] or secreted by the cells under investigation). The cloning of Fn14-ΔEC afforded us the opportunity to directly test whether ectopic Fn14 overexpression promoted TWEAK-independent Fn14 signaling. As our readout for Fn14 signaling we selected NF-κB pathway activation, since TWEAK:Fn14 engagement in diverse cell types has been shown to stimulate this intracellular pathway (reviewed in [4]). HEK293/NF-κB-luc reporter cells were transiently transfected with vector or expression plasmids encoding either Fn14-FL (our positive control), Fn14-ΔEC, or Fn14-ΔCT, an Fn14 mutant that is missing the C-terminal 18-aa residues of the 28-aa Fn14 cytoplasmic tail (Fig. 3A) and therefore cannot bind TRAFs and activate signaling pathways (our negative control) [44]. All Fn14 proteins had an N-terminal Myc epitope tag. Cells were harvested 24 h later and processed for Western blot analysis and luciferase reporter assays. The three Fn14 proteins were expressed at similar levels (Fig. 3B). We noted previously that Fn14 does not migrate at its predicted molecular mass when analyzed by SDS-PAGE under reducing conditions [1] and this is again illustrated on this particular blot. For example, the predicted molecular mass (MM) of the mature, 112-aa myc-tagged Fn14-FL protein is 12.1-kDa, but its apparent MM on this gel is ∼23-kDa. Also, the 101-aa Fn14-ΔCT mutant (11.0-kDa predicted MM) is running at a lower apparent MM than the much smaller, 77-aa Fn14-ΔEC mutant (8.4-kDa predicted MM). The basis for these findings is not known but protein amino acid composition and isoelectric point are two interrelated factors that can influence the mobility of proteins on SDS polyacrylamide gels. For small proteins like Fn14, the effects of these factors on electrophoretic mobility are even more apparent. One possible explanation for the anomalous migration of the three Fn14 species relative to one another is that the Fn14-FL and Fn14-ΔEC proteins are acidic (predicted pIs of 5.8 and 5.3, respectively) while the Fn14-ΔCT mutant is basic (predicted pI of 8.8).

Bottom Line: Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions.Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122.These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

Show MeSH
Related in: MedlinePlus